A novel generation 1928zT2 CAR T cells induce remission in extramedullary relapse of acute lymphoblastic leukemia

Abstract

Background: Anti-CD19 chimeric antigen receptor (CAR) T cells have shown promise in the treatment of B cell acute lymphocytic leukemia (B-ALL). However, its efficacy in B-ALL patients with extramedullary involvement is limited due to poor responses and neurotoxicity. Here, we utilized a third generation of CAR T cell vector, which contains the Toll/interleukin-1 receptor (ITR) domain of Toll-like receptor 2 (TLR2), to generate 1928zT2 T cells targeting CD19, and evaluated the efficacy of 1928zT2 T cells in relapse or refractory B-ALL patients with extramedullary involvement.

Methods: 1928zT2 T cells were generated by 19-28z-TLR2 lentiviral vector transfection into primary human T lymphocytes. The anti-leukemia effect of 1928zT2 T cells were determined by killing assays and in xenografts. Three patients diagnosed as relapse or refractory ALL with extramedullary involvement were infused with 1928zT2 T cells, and the clinical responses were evaluated by BM smear, B-ultrasonography, PET/CT, histology, flow cytometry, qPCR, ELISA, and luminex assay.

Results: 1928zT2 T cells exhibited enhanced effector function against CD19+ leukemic cells in vitro and in a xenograft model of human extramedullary leukemia. Notably, the 1928zT2 T cells eradicated extramedullary leukemia and induced complete remission in the three relapse and refractory ALL patients without serious adverse effects. 1928zT2 T cells expanded robustly in the circulation of these three patients and were detected in the cerebrospinal fluid of patient 3. These three patients experienced cytokine release syndrome (CRS) with grade 2 or 3, which remitted spontaneously or after tocilizumab treatment. None of the three patients suffered neurotoxicity or needed further intensive care.

Conclusions: Our results demonstrate that 1928zT2 T cells with TLR2 incorporation augment anti-leukemic effects, particularly for eradicating extramedullary leukemia cells, and suggest that the infusion of 1928zT2 T cells is an encouraging treatment for relapsed/refractory ALL patients with extramedullary involvement.

Trial registration: ClinicalTrials.gov identifier NCT02822326 . Date of registration: July 4, 2016.

Keywords: Acute lymphocytic leukemia (ALL); Chimeric antigen receptor (CAR) T cells; Extramedullary ALL; Relapsed and refractory; TLR2.

Conflict of interest statement

Ethics approval and consent to participate

This study was approved by the Human Ethics Committees of the Guangdong General Hospital. The patient and donor gave their written informed consent in accordance with the Declaration of Helsinki. This study is registered at www.ClinicalTrials.gov as NCT02822326. All animal experimental protocols were performed in accordance with instruction guidelines from the China Council on Animal Care and approved by the guidelines of the Ethics Committee of Animal Experiments at Guangzhou Institutes of Biomedicine and Health (GIBH).

Consent for publication

The authors have obtained consent to publish from the participant to report individual patient data.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1

The 1928zT2 T cells presented elevated killing percentage of CD19+ cells compared with 1928zT cells in vitro. The killing assay with co-cultured 1928zT2 T cells and CD19-expressing NALM6 cells revealed that the 1928zT2 T cells lysed robustly the NALM6 cells at varied indicated ratios of E:T, in particular still eradicated more than 75% NALM6 cells at low ratios, whereas the 1928zT cells presented less than 50%. Similar data were observed in the K562 cells with CD19 expression, which were also utilized to evaluate the anti-CD19+ leukemia effect of 1928zT2 T cells. The K562 cells without CD19 expression were used as negative controls and virtually no killing effect was observed in 1928zT cells and GFP-control T cells

Fig. 2

The 1928zT2 T cells promoted the regression of subcutaneous leukemia ex vivo. a The cell line-derived xenografts were established in the immunodeficient NSI mice by subcutaneous injection of 2 × 105 NALM-6 cells. These mice received one infusion of 2 × 106 GFP T, 1928z T cells, or 1928zT2 T cells, respectively. b BLI results revealed a marked tumor mass grew subcutaneously in the GFP-T group and similar signal was seen in the 1928z T cells group. However, the 1928zT2 T cells group exhibited significantly reduced signal compared to the other groups, indicating that tumors were significantly suppressed in these mice. c The tumor was extracted, and the specimen were showed. The tumor mass was smallest in the mice treated with 1928zT2 T cells, presenting as the lowest specific weight of tumor mass

Fig. 3

Small dose of 1928zT2 T cell infusion eradicated leukemia and induced CR in patient 1. a The diagram shows the development and therapeutic process of this ALL patient with extramedullary involvement. The 34-year-old female patient was diagnosed as B-ALL (CD19+, BCR/ABL-) in April, 2015, received allo-HSCT in November, 2015, and had a relapse in extramedullary (EM) tissues in August, 2016. She received fludarabine (F) and cytarabine (C) before cells infusion. Forty-six days after 1928zT2 T cells infusion (as low as 5 × 104 cells/kg), the patient achieved CR and maintained remission in the follow-up. VDLCP, vincristine, daunomycin, cyclophosphamide, asparaginase, and dexamethasone; Hyper-CVAD A, cyclophosphamide, vincristine, doxorubicin, and dexamethasone; SC, systemic chemotherapy; b PET/CT data showed obviously an abnormal intense high metabolic mass in the left breast. Restage of PET/CT on day 30 after cells infusion presented that the lesion became hypometabolic state and no abnormal signal was observed thereafter. c The histological results showed the infiltration of megakaryocytes, erythroblasts, and myeloid cells in the tumor section, proven to be extramedullary relapse. d B-mode ultrasound showed an inhomogeneous hypo-echoic mass about 2.8 × 1.6 cm in diameter before cells infusion and reduction of mass size with 2.3 × 1.1 cm on day 14. The abnormal hypo-echoic mass was disappeared on day 46 and thereafter

Fig. 4

Infusion of 1928zT2 T cells induced complete remission in BM and extramedullary relapse in patient 2. a The 15-year-old male was diagnosed as B-ALL in October, 2014. He had the first recurrence (RE1) in CNS, and the leukemia recurred again with BM and extramedullary involvement (RE2). After 3-day usage of F and C, he received 1928zT2 T cells infusion (5 × 105 cells/kg) and got CR 1 month later. Particularly, he underwent allo-HSCT again in July, 2017 and maintained remission during the follow-up. b The BM smear image revealed typical leukemia blasts at baseline disappeared 1 month after 1928zT2 T cells infusion. c The PET/CT revealed that an abnormal low density area and corresponding intense high metabolic region beneath the kidney capsule (red arrow). Restage of PET/CT after cells infusion presented that no low density area in the kidney, whereas there were diffuse high metabolic distribution with increased SUV due to the metabolism of 18F-FDG in the kidney

Fig. 5

The 1928zT2 T cells exhibited potent anti-leukemia effect in patient 3. a shows the development and therapeutic process of this ALL patient with CNS-3 status and extramedullary involvement. He was diagnosed as ALL in March, 2000. The first recurrence (RE1) involved in BM and CNS, and the second RE2 occurred in CNS again. The third relapse (RE3) was identified in CNS and EM in June, 2017. This patient also received fludarabine (F) and cytarabine (C) daily to achieve lymphodepletion and a split-dose infusion of 1928zT2 T cells (1 × 106 cells/kg) was applied. b of the PET/CT data shows multiple organs involved by extramedullary ALL, including almost the entire pleura, peritoneum, pelvic fascia, pancreas, and multiple lymph nodes. Restage of PET/CT on D60 after cells infusion presented that no infiltration of cancer cells into the involved tissues at all. c A biopsy of the right-sided supraclavicular node mass at baseline shows the presence of blasts, megakaryocytes, erythroblasts, and myeloid cells, proven to be extramedullary relapse. d of B-mode ultrasound on tissues below xiphoid shows an inhomogeneous hypo-echoic mass about 3.0 × 1.9 cm in diameter before cells infusion and disappearance of the mass during the follow-up. e shows the typical presence of leukemic blast cells (at least 5 blasts in the whole cytocentrifuged smear) in CSF at baseline (Pre-treatment) and only normal mature lymphocytes was seen at D18 and thereafter. f of the flow-cytometric data shows that despite about 20.8% blasts with CD19+CD10+ phenotype was observed in the WBC from CSF before cells infusion, there were undetectable levels of blasts at several time points after treatment. g shows that 50% of the cerebrospinal cells were identified as XY-type recipient cells by FISH, indicating the relapse in the CSF. However, there were 100% recipient cells (XX) in CSF after 1928zT2 T cells infusion, suggested complete eradication of blasts in CSF

Fig. 6

The expansion of 1928zT2 T cells and relevant clinical changes in these patients. a The results of flow-cytometric GFP-positive cells analysis in patient 1 showed the 1928zT2 T cells were expanded significantly which began on day 5, peak on day 12, and disappeared after day 92. The CD19-positive cells in periphery blood were dropped quickly within 10 days to the undetectable level and increased again. b Patient 2 presented with the presence and elevation of GFP+1928zT2 T cells in the periphery blood from day 10 to day 50. The CD19+ cells were reduced significantly till undetectable. c Since the patient 2 had the relapse also in the BM, we found the upregulation of 1928zT2 T cells in the BM, accompanied with eradication of CD19+ cells. d The 1928zT2 T cells was expanded significantly on day 12 with 57% of T cells and was undetectable after day 17 in the patient 3. e shows the presence of 1928zT2 T cells in CSF from patient 3 with a peak on day 18, indicating the trafficking ability of these cells. Consistently, the blast cells in CSF were undetectable after day 18. f shows changes in body temperature after1928zT2 T infusion in these three patients. g Patient 1 suffered from grade 2 CRS with fever from day 7 to day 13. IL-6 was elevated slightly with a peak level on day 11, coincided with the occurrence of CRS. The C-Reactive protein (CRP) was also increased. h Patient 2 also suffered from CRS with grade 2 from day 9 to day 12 and the expression pattern of IL-6 and CRP was similar to patient 1. i For the patient 3, IL-6 was elevated markedly with a peak level on day 10 and decreased after twice usage of tocilizumab, coincided with the occurrence of CRS from day 7 to day 15 (black arrows)

Fig. 7

The change of routine blood test and serum cytokines in these patients before and after infusion of 1928zT2 T cells. a–c The circulating white blood cells count (WBC), absolute neutrophil count (ANC), absolute lymphocyte count (ALC), and platelet count (PLT) increased after treatment, indicating the recovery of routine blood test results. These blood test maintained in a relatively stable level during the follow-up in these three patients. d The qPCR data revealed the expression pattern of the cell adhesion and pro-inflammatory genes in patient 2. e shows the cytokine levels in the serum from patient 3 at the indicated time points after1928zT2 T cells infusion. The expressions of IL-22, MCP-1, and HGF presented as the same pattern with IL-6. The IFN-α, IL-17A, GM-CSF, IL-1β, IL-12, IL-7, and IL-15 were below the limits of detection at indicated time points and the other 34 cytokines showed little changes